Objectives To identify the locus of trophoblast-specific and cAMP-responsive elements in the rhesus monkey growth hormone variant gene. Transcriptional activation of the rhesus monkey growth hormone-variant (mGH-V) gene in syncytiotrophoblasts (STB) is developmentally regulated by as yet undefined trophoblast-specific and cAMP-responsive mechanisms. Pituitary growth hormone transcription depends heavily on the expression of the POU domain transcription factor Pit-1, but although expression of this factor has recently been detected in the primate placenta, no role for Pit-1 in the regulation of mGH-V transcription has been defined. Examination of Pit-1-like elements in the mGH-V promoter region have not led to the identification of any bound Pit-1 through competitive mobility shift or supershift analysis. Systematic mutational analysis of the mGH-V promoter also did not demonstrate a critical functional role for either of the conserved Pit-1 motifs in transcription in placental or non-placental cells. Other transcription factors, however, have been identified which interact with the mGH-V promoter in choriocarcinoma cell lines and primary STBs. Sp1 was demonstrated by supershift analysis to bind to a consensus sequence (-136/-131) within the mGH-V promoter. Mutation of this motif, however, only resulted in a 2 fold decrease in transcription in STBs. AP2 also was shown to bind to the consensus TSE element at -162/-154. The AP2/TSE complex, and its corresponding supershift, was only detected with placental cell line nuclear extracts (JEG-3 and BeWo, not with HeLa or Cos). Mutational analysis of this AP2/TSE motif will examine its role in mGH-V transcription in both placental and non-placental cells. Deletional analysis of the mGH-V promoter demonstrating little loss in cAMP-responsive transcriptional activity corresponding to this region, however, implies only a supportive role for AP2 in mGH-V transcription Transient transfections of reporter plasmids containing systematic 10 base mutations throughout the mGH-V promoter indicated that cAMP-stimulated activity originates in two regions, -140/-111 and -72/-53. Mutations in either of these regions resulted in up to 30 fold losses in activity in STB cell and JEG-3 choriocarcinoma cell line cultures. Other than Sp1, we have not detected interactions of any known factors within these regions of the mGH-V promoter. Therefore, the factors primarily responsible for the trophoblast-specific and cAMP responsive transcription of mGH-V have yet to be defined. We have identified a novel binding activity in JEG extracts corresponding to the critical -140/-111 region of the promoter and will investigate its role in the trophoblast specific expression of mGH-V. Key words placenta, gene transcription, placental growth hormone, transcription factors